The field of this invention is lignin and lignin modifying and degrading enzymes. Lignin is a complex polymer formed by free radical polymerization of substituted cinnamyl alcohol precursors. Lignin constitutes 15-35% of dry wood weight. During pulping processes, cellulosic fibers must be liberated from their encasing lignin matrix so that they can associate with one another, yielding strength in the final product. This polymer separation can be accomplished by removal of lignin as in chemical pulps, or by maintaining the lignin as in high yield mechanical pulps. During the bleaching process, lignin is removed and the resulting pulp is brightened.
Lignin is highly resistant to biological attack. No organism has been demonstrated to grow on lignin as the sole carbon source. The complex lignin polymer, however, is completely degraded by pure cultures of various higher order fungi. The most extensive physiological investigations of lignin biodegradation by white-rot fungi have been conducted with a single member of the family Corticraceae, Phanerochaete chrysosporium Burds.
Under defined laboratory conditions, fungal lignin degradation is not observed during approximately the first 3 days of culture. Subsequently, the culture becomes starved for carbon or nitrogen, and lignin degradation is first observed at day 4 or 5 of culture and is maximal at 6 days. The induction of lignin degradation in response to carbon and nitrogen starvation indicates that fungal lignin metabolism is a secondary metabolic event (Keyser, P., Kirk, T. K. and Zeikus, J. G. [1978] J. Bacteriol. 135:790-797).
Fungal lignin degradation currently is commercially impractical for several reasons. The rate of lignin degradation is unacceptably slow since ligninolytic activity is induced only as a result of secondary metabolism. Therefore, a period of initial growth followed by starvation is required. Furthermore, fungi metabolize cellulosic fibers as their primary food source, resulting in reduced pulp yield and an inferior pulp product.
Tien and Kirk have disclosed a preparation capable of oxidatively cleaving C.sub..alpha. -C.sub..alpha. bonds in lignin model compounds and oxidatively converting veratryl alcohol to veratrylaldehyde (Tien, M. and Kirk, T. K. [1984] Proc. Natl. Acad. Sci. 81: 2280-2284) . This preparation displays on an SDS-polyacrylamide gel predominantly one protein with an apparent molecular weight of 42 kD and several minor bands. Thus the preparation is a mixture of proteins. Subsequent to the publication of this paper, several scientific papers were published disclosing an inability to isolate the major protein from the mixture. These articles are as follows: Huynh, V-B and Crawford, R. L. (1985) FEMS Microbiology Letters 28:119-123; Leisola, M. et al. (1985) Lignin Biodegradation Workshop; and Gold, M. H. et al. ( 1985) Lignin Biodegradation Workshop.
These attempted protein isolations have been done either by ion-exchange chromatography or by size exclusion ion exchange column chromatography. The fractions containing the indicated component have been analyzed by isoelectric focusing or SDS-polyacrylamide gel electrophoresis, and have been shown to contain more than one protein species.
In pending U.S. application Ser. No. 845,655, filed on Mar. 28, 1986, now U.S. Pat. No. 4,687,741, there is disclosed and claimed, inter alia, an enzyme preparation designated rLDM.TM. 6. (rLDM.TM. is the trademark of the Repligen Corporation, Cambridge, Mass., for lignin degrading and modifying enzymes.) This preparation is substantially pure and hence also substantially free of other rLDM.TM. and degrading native proteases. It is also substantially free of native proteins having different activities which are present in the Tien and Kirk mixture, supra. Advantageously, this rLDM.TM. 6 preparation possesses desirable properties for use in pulping wood and treating effluent which the Tien and Kirk preparation does not have. Specifically, the Tien and Kirk mixture has a lower specific activity than rLDM.TM. 6. The rLDM.TM. have been characterized by the critical property of being able to catalyze the oxidation of veratryl alcohol to veratrylaldehyde (ligninase activity), and the following physical parameters:
(1) molecular weight as determined by SDS-PAGE; PA1 (2) amino acid composition; PA1 (3) heme content; PA1 (4) homology by antibody reactivity; PA1 (5) specificity of activity against lignin model substrates; and PA1 (6) elution from a FPLC column at specified sodium acetate molarities.
Though the preparation of rLDM.TM. 6 can be carried out as described in Ser. No. 845,655, now U.S. Pat. No. 4,687,741, the disclosure of which is incorporated herein by reference, it could be expected that more efficient production of rLDM.TM. 6 would be realized if DNA encoding rLDM.TM. 6 could be cloned and expressed in a suitable host. The task of such a successful cloning and expression is formidable. Further, it is nonobvious (unpredictable) whether there can be expression of activity of a P. chrysosporium gene in a heterologous host since this has never been achieved previously. Still further, the prior art provides no suggestions as to how such a cloning and expression invention could be achieved. The applicants in this application for patent have overcome these prior art barriers by successfully cloning and expressing rLDM.TM. 6.